Killer T cell receptor recognizing human immunodeficiency virus

ABSTRACT

The present invention relates to a polypeptide which is a constituent of a killer T cell receptor and is capable of injuring human immunodeficiency virus-infected cells; a DNA encoding this polypeptide; a recombinant vector comprising this DNA and a vector; a transformant obtained by transferring this recombinant vector into a host cell; a process for producing the above polypeptide characterized by culturing the above transformant in a medium, thus forming and accumulating the polypeptide in the culture and then recovering the polypeptide from the culture; an antibody reacting specifically with the above polypeptide; human-mouse type killer T cell receptor αchain and βchain in each of which the variable region site of the above polypeptide is sustained while the constant region site thereof has been replaced by that of the human type; transgenic animals having the above polypeptide expressed therein; and anti-HIV agents containing the above polypeptide.

TECHNICAL FIELD

The present invention relates to a polypeptide which is a constituent ofa killer T cell receptor capable of specifically injuring humanimmunodeficiency virus-infected cells; a DNA encoding this polypeptide;a vector containing the DNA; a transformant obtained by transferring thevector into a host cell; a process for producing the polypeptide whichis a constituent of a T cell receptor; transgenic animals having thepolypeptide expressed therein; antibodies reacting specifically with thepolypeptide; and anti-HIV agents containing the polypeptide which is aconstituent of the killer T cell receptor.

BACKGROUND ART

Recently, there have been reports on the importance of a CD8molecule-positive killer T cell involved in the initial phylaxis [Koup,R. A. et al., Nature, 370, 416 (1994)], delay in the development of AIDS[Levy, J. A. et al., Immunol. Today, 17, 217 (1996)] and resistance tothe infection [Rowland-Jones, S. et al., Nature Med., 1, 59 (1995)] ofhuman immunodeficiency virus (HIV), and further the HIV suppressiveability of a humoral factor secreted by the killer T cell [Cocchi, F. etal., Science, 270, 1811 (1995); Baier, M. et al., Nature, 378, 563(1995)] have been reported.

Ho et al., reported that as a result of tracing with the viral loads ofHIV-infected individuals and the immune responses induced by the viruswith the elapse of time, the virus temporarily increased in vivo afterinfection but rapidly decreased as CD8⁺ killer T cell precursor specificto the virus (cytotoxic T lymphocyte precursor; hereinafter referred toas CTL-p) appeared; and 6 to 8 weeks after that most of the viruses werecleared virus-specific IgG antibodies appeared. Thus the study suggestedthe importance of the cell-mediated immunity mainly with CD8⁺ killer Tcells in the initial phylaxis [Nature, 370, 416 (1994)].

Then, several articles reported that the presence of asymptomaticpatients whose CD4 T cell counts have not decreased over ten and severalyears and who have not developed AIDS, and in these patients thecell-mediated immunity, in which CD8 positive T cells and Th1 typehelper T cells are mainly involved, is dominant in vivo over the humoralimmunity, and CD8 positive T cells secreting MIP-1 α, β [Science, 270,1811 (1994)] or IL-16 [Nature, 378, 563 (1995)], capable of suppressingthe proliferation of HIV, were identified. Thereafter the importance ofthe CD8 positive T cells including killer T cells and the cell-mediatedimmunity in the initial phylaxis and in the protection of thedevelopment of AIDS is increasingly noticed [Immunol. Today, 17, 217(1996)].

The invasion of HIV into cells is, for example on T cells, regulated byfusin on the cell surface [Feng, Y. et al., Science, 272, 872 (1996)],and on macrophages, regulated by chemokine receptors, i.e., CC-CKR-5[Deng, H. et al., Nature, 381, 661 (1996); Drajic, T. et al., Nature,381, 667 (1996)]. It was reported that chemokines, i.e., MIP-1 α, β, orIL-16, binding specifically to a variety of chemokine receptors, inhibitthe invasion of HIV into cells [Cocchi, F. et al., Science, 270, 1811(1995); Bleul, C. C. et al., Nature, 382, 829 (1996)]. It was alsoreported that HIV-invasive sites are chemokine receptors, i.e., CC-CKR-4or CC-CKR-5 [Science, 272, 872 (1996); Nature, 381, 661 (1996)] based onthe fact that human races congenitally having a deletion in geneCC-CKR-5 escape from being infected with HIV [Nature, 382, 722 (1996);Cell, 86, 367 (1996)]. That is, it has been found that factors, e.g.,MIP-1 α, β, and RANTES released by CD8⁺CTL, block chemokine receptors soas to obstruct the invasion of HIV into cells, thereby suppressing theintracellular increase of HIV.

Moreover, it was shown that a part of the virus that invades via achemokine receptor into a cell is HIV envelope protein gp160 V3 region[Nature, 384, 179 (1996); Nature, 384, 184 (1996)]. It is said that theHIV envelope protein gp160 V3 region determines the type of a cell,tropism infected with virus. Particularly, in a mouse, Env-K1 (or18IIIB:RIQRGPGRAFVTIGKP18)[Takahashi, H. et al., Proc. Natl. Acad. Sci.USA, 85, 3105(1988)], the amino acid sequence 315 to 329 in the HIVenvelope protein gp160 V3 region derived from HIV IIIB strain ispresented on the cell surface together with Class I MHC molecule(D^(d)), and recognized by a specific killer T cell receptor [Takahashi,H. et al., J. Exp. Med., 170, 2023 (1989)]. At the same time, Env-K1 ispresented on the cell surface together with Class I MHC molecules,HLA-A2, HLA-A3 and the like, which are recognized relatively widely inhuman, and the in vivo presence of killer T cells recognizing Env-K1 isconfirmed in HIV-infected individuals [Clerici, M. et al., J. Immunol.,146, 2214 (1991); Dadaglio, G. et al., 147, 2302 (1991)].

When vaccinia virus recombined with HIV envelope (gp160) gene wasinoculated in vivo into a healthy individual, killer T cells recognizingspecifically ENV-K1 presented as an antigen by a variety of HLAs wereinduced, and the killer T cells specifically injured self-cells infectedwith the gp160 recombinant vaccinia virus [Achour, A. et al, FifthInternational Conference on AIDS, p.546 (Abstract) (1989)]. However,killer T cell clones have not been produced.

Further, the V3 region within envelope gp160 including Env-K1 is knownto be the recognition site of a neutralization antibody specific to HIV[Palker, T. J. et al., Proc. Natl. Acad. Sci. USA, 85, 1932 (1988);Rusche, J. R. et al., Proc. Natl. Acad. Sci. USA, 85, 3198 (1988);Goudsmit, J. et al., Proc. Natl. Acad. Sci. USA, 85, 4478 (1988)] or therecognition site of a helper T cell [Takahashi, H. et al., J. Exp. Med.,111, 579 (1990); Clerich, M. et al., Nature, 339, 383 (1989); Takeshita,T. et al., J. Immunol., 154, 1973 (1995)].

An anti-V3 antibody has a neutralizing activity against HIV. However,the anti-V3 antibody must be administered in vivo in a large quantity tosuppress the proliferation of HIV. On the other hand, since an antibodyis a macromolecule, such mass administration is undesirable. Therefore,establishing the killer T cell clone, which specifically recognizes V3,especially Env-K1, and detailed investigations of the molecularstructure of the T cell receptor are considered to be useful indeveloping next generation agents for inhibiting the invasion of thevirus by blocking the invasion of HIV.

Accordingly, it has been expected for the analysis of the HIV-specifickiller T cell clone and for the development of a transgenic animal as anindividual to express the functional receptor gene of such killer T cellto bring information extremely useful in treatment and researches forAIDS. However, so far neither such development nor analysis has not beenreported.

It is required that HIV specific killer T cells be used in searching thefate of human immunodeficiency virus, and in developing treatment andpharmaceuticals for AIDS. Further it is also required to investigate howthe previous expression of the gene can have an effect on the preventionof the infection, and how shutting the virus in, in which the gene areexpressed after infection, can have an effect on the treatment. That is,there is a desire to analyze the CD8 positive killer T cell clonespecifically injuring human immunodeficiency virus-infected cells and todevelop a transgenic animal expressing the killer T cell receptor.

DISCLOSURE OF THE INVENTION

The present invention relates to (1) to (17) as shown below.

(1) A polypeptide which is a constituent of a killer T cell receptor andis capable of injuring specifically human immunodeficiencyvirus-infected cells;

(2) The polypeptide according to the above (1) wherein the humanimmunodeficiency virus is HIV-1;

(3) The polypeptide according to the above (2) wherein the HIV-1 isHIV-1 IIIB;

(4) The polypeptide according to any one of the above (1) to (3),wherein a polypeptide constitutes a killer T cell receptor whichrecognizes specifically human immunodeficiency virus envelope proteingp160.

(5) The polypeptide according to the above (4) wherein the recognitionregion of the killer T cell receptor which recognizes specifically humanimmunodeficiency virus envelope protein gp160 is a V3 region of thegp160;

(6) The polypeptide according to the above (5) wherein the recognitionregion is a region comprising the amino acid sequence 315 to 329 in thehuman immunodeficiency virus envelope protein gp160 V3 region;

(7) A polypeptide which comprises an amino acid sequence shown in SEQ IDNO: 7 or 9, or a polypeptide, which comprises an amino acid sequencewherein one or more of amino acids in the amino acid sequence aresubstituted, deleted or added, and is capable of injuring specificallyhuman immunodeficiency virus infected-cells.

The above-mentioned substitutions, deletions or additions of one or moreof amino acids can be performed by means of a well-known art before thefiling of the present applicaiton, the site-directed mutagenesis method.In addition, the term “one or more of amino acids” used herein means thenumber of amino acids which can be substituted, deleted, or added by thesite-directed mutagenesis method.

The polypeptide which comprises an amino acid sequence wherein one ormore amino acids are substituted, deleted, or added, and is capable ofinjuring specifically human immunodeficiency virus-infected cells can beprepared according to the methods described in Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989) (hereinafter abbreviated as Molecular Cloning ₂nd ed., ), CurrentProtocols in Molecular Biology, Supplement 1 to 38, John Wiley & Sons(1987-1997) (hereinafter abbreviated as Current Protocols in MolecularBiology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad.Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research,13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488(1985), Proc. Natl.Acad. Sci. USA, 81, 5662 (1984), Science, 224, 1431(1984), PCTWO85/00817(1985), Nature, 316, 601(1985) and the like.

(8) A DNA encoding the polypeptide according to any one of the above (1)to (7).

(9) The DNA having the nucleotide sequence shown in SEQ ID NO:6 or 8.

(10) A DNA which encodes the polypeptide, capable of injuringspecifically human immunodeficiency virus-infected cells, which canhybridize with the DNA according to the above (8) or (9) under stringentconditions.

As used herein, the term “the DNA which encodes the polypeptide, capableof injuring specifically human immunodeficiency virus-infected cells,which can hybridize under stringent conditions” means a DNA which can beobtained by using the DNA of the above (8) or (9) as a probe accordingto the colony hybridization technique, the plaque hybridizationtechnique or the southern blot hybridization technique or the like. Forexample the DNA can be identified by performing hybridization using afilter, to which DNA derived from a colony or a plaque is immobilized,under the presence of 0.7 to 1.0M NaCl at 65° C. and then by washing thefilter using 0.1-2×SSC (saline-sodium citrate) solution (where thecomposition of 1×SSC solution is 150 mM sodium chloride, 15 mM sodiumcitrate) at 65° C.

Hybridization can be performed according to the methods shown inprotocols including Molecular Cloning 2^(nd) ed., Current Protocols inMolecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach,Second Edition, Oxford University Press (1995), and the like.

The DNA which can be hybridized is, for example, a DNA having homologyof 80% or more, preferably 95% or more, to the nucleotide sequence shownin SEQ ID NO: 6 or 8.

(11) A recombinant vector comprising the DNA according to any one of theabove (8) to (10) and a vector.

(12) A transformant obtained by introducing the recombinant vectoraccording to the above (11) into a host cell.

(13) A process of producing the polypeptide according to any one of theabove (1) to (7), which comprises culturing the transformant of theabove (12) in a medium, forming and accumulating the polypeptide of anyone of the above (1) to (7) in the culture, and recovering thepolypeptide from the culture.

(14) An antibody which specifically reacts with the polypeptideaccording to any one of the above (1) to (7).

(15) The polypeptide according to any one of the above (1) to (7),having a human type constant region site.

(16) Transgenic animals, having the polypeptide according to any one ofthe above (1) to (7) expressed therein.

(17) Anti-HIV agents containing the polypeptide according to any one ofthe above (1) to (7).

The killer T cell clone injuring HIV-infected cells can be establishedby preparing antigens, administering the antigens to animals forimmunization, removing sensitized lymphocytes from the cells of theimmunized animals and stimulating the sensitized lymphocytes.

As the HIV strain, which is used for producing the killer T cell cloneinjuring HIV infected cells, includes HIV-1 III3 strain or the like canbe mentioned. As the epitope, Env-K1 containing the amino acid sequence315 to 329 presented in V3 region within HIV-1 envelope protein gp160[amino acid sequence; RIQRGPGRAFVTIGK (Takahashi, H. et al., Proc. Natl.Acad. Sci. USA, 85, 3105 (1988), hereinafter referred to as P18) can bementioned.

The methods for administering the antigen include the following: amethod using ISCOM (Immunostimulating complex) which is a specialimmunopotentiating substance (adjuvant) [Takahashi, H. et al., Nature,344, 873 (1990)]; a method using a complex of QS-21, one of constituentof ISCOM, and HIV envelope protein gp160 [Wu, J. et al., J. Immunol.,148, 1438(1992)]; a method using a recombinant vaccinia virus whereinthe gp160 gene is introduced [Takahashi, H. et al., Proc. Natl. Acad.Sci. USA, 85, 3105 (1988)] and a method using self-dendritic cell formedby binding, a self-cell, into which the gp160 gene is introduced, andEnv-K1 [Takahashi, H. et al., Int. Immuno., 5, 849 (1993)] since it isknown to be difficult for general purified protein antigens and the liketo induce the killer T cells.

Example of animals for immunization includes mice, rats, rabbits,monkeys and the like. For example, the mice for immunization havevarious genetic characters such as B10.PL(H-2^(u)), B10.P(H-2^(p)),B10.Q(H-2^(q)), and B10.A(H-2^(a)). In particular, a BALB/c(H-2^(d))mouse which shows a high reactivity with P18 is preferred.

Sensitized lymphocytes are obtained by removing the spleen from theimmunized animal, and performing a treatment such as the removal oferythrocytes. To stimulate the sensitized lymphocytes,antigen-presenting cells, fibroblasts and the like, which expressantigens or to which antigens are bound, are irradiated with radiationor treated with mitomycin-C are used. These cells are preferably thesame type of cell line as the immunized cells. P18 specific killer Tcell clone can be established by stimulating continuously with thecells. The killer T cell clones injuring HIV infected-cells according tothe present invention include RT-1, RT-2, RT-3 and the like. A methodfor confirming T cells is, e.g., FACScan using an antibody to amolecular marker expressed on the cell.

A T cell αβreceptor is a heterodimer protein formed by disulfide bondsof αchain and βchain polypeptides. The receptor forms a complex with CD3and is expressed on the surface layer of a T cell. The specific T cellaαβreceptor comprises many different V-(D)-J-C regions. The type of thereceptor itself is considered to be defined according to the amino acidsequence of V region and the specificity to a foreign matter accordingmainly to the amino acid sequences of D and J regions. Accordingly, theT cell receptor gene is identified from P18-specific killer T cell cloneby determining V regions for T cell receptor αchain and βchain, and byidentifying the entire gene sequence.

The V regions of the T cell receptor αchain and βchain are identified bypolymerase chain reaction (hereinafter referred to as PCR) with primersproduced based on the sequences of the obtained mRNA and of each Vregion. Then the reverse transcription-PCR (RT-PCR) is performed for theobtained mRNA to produce cDNA. Thus the sequence can be determined.

The full-length DNA having a junctional region specific to p18 isproduced by the recombinant PCR technique to determine the whole genesequence.

The total RNA is prepared from the T cell clone by the guanidinethiocyanate—cesium trifluoroacetate method [Methods in Enzymology, 154,3 (1987)], acidic guanidine thiocyanate-phenol-chloroform (AGPC) method[Analytical Biochemistry, 162, 156 (1987), Experimental Medicine 9, 1937(1991)] and the like.

From the total RNA mRNA is prepared as poly (A)⁺RNA according to themethod using the oligo(dT) immobilized cellulose column technique(Molecular Cloning 2^(nd) ed., ), the method using an oligo dT latex,and the like.

Alternatively, mRNA can be prepared directly from tissues or cells byusing Fast Track mRNA Isolation Kit (manufactured by Invitrogen), QuickPrep mRNA Purification Kit (manufactured by Pharmacia), and the like.

From the total RNA or mRNA obtained, cDNA libraries are obtained byusing conventional method.

For example the cDNA library can be prepared according to the methoddescribed in Molecular Cloning 2^(nd) ed., Current Protocols inMolecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach,Second Edition, Oxford University Press (1995) and the like, or by usingcommercially available kits, such as, SuperScript Plasmid System forcDNA Synthesis and Plasmid Cloning (manufactured by Gibco BRL) andZAP-cDNA Synthesis Kit (manufactured by STRATAGENE).

As the cloning vectors for preparing the cDNA library, any of phagevectors and plasmid vector can be used so long as it is capable ofautonomously replicating in Escherichia coli K12.

Examples of suitable vectors are ZAP Express [manufactured bySTRATAGENE, Strategies, 5, 58 (1992)], pBluescript II SK(+) [NucleicAcids Research, 17, 9494 (1989)], Lambda ZAP II (manufactured bySTRATAGENE), λ gt10, λ gt11 [DNA Cloning, A Practical Approach, 1, 49(1985)], λ TriplEx (manufactured by CLONTECH), λ ExCell (manufactured byPharmacia), pT7T318U (manufactured by Pharmacia), pcD2 [Mol. Cell.Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], and pAMo[J. Biol.Chem., 268, 22782-22787 (1993), another name, pAMoPRC3Sc(JP-A-05-336963)].

Any microorganism belonging to Escherichia coli can be used as a hostmicroorganism. Examples of the host microorganisms are Escherichia coliXL1-Blue MRF' [manufactured by STRATAGENE, Strategies, 5, 81 (1992)],Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088[Science, 222, 778 (1983)], Escherichia coliY1090 [Science, 222, 778(1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)],Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coliJM105 [Gene, 38, 275 (1985)], Escherichia coli SOLR™ Strain(manufactured by STRATAGENE), and Escherichia coli LE392 (MolecularCloning 2^(nd) ed.,).

In addition to the cDNA library constructed by the above-mentionedmethods, commercially available cDNA library can be used.

From the cDNA library constructed by the above-mentioned methods, thecDNA clone containing the DNA of the present invention can be selectedthe colony hybridization, or the plaque hybridization [Molecular Cloning2^(nd) ed.,] using probes labeled with isotope or fluorescence.

The probes can include a fragment obtained by amplifying a part of cDNAusing PCR [PCR Protocols, Academic Press (1990)] with primers based on apartially known nucleotide sequence, and an oligonucleotide based on apartially known nucleotide sequence.

The primer prepared based on such sequences can be employed when bothnucleotide sequences of the full-length cDNA on the 5′-end side and3′-end side are known in sequences such as ESTs,.

cDNA is synthesized from the mRNA using the cDNA clone having the DNA ofthe present invention selected as described above, according to theabove techniques.

By the use of 5′-RACE (rapid amplification of cDNA ends) and 3′-RACE[Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)] wherein PCR is conductedwith primers based on a nucleotide sequence of an adapter which is addedto both ends of the cDNA and with those based on a partially knownnucleotide sequence, cDNA which is upstream (5′-end side) and downstream(3′- end side) from the amplified fragment can be obtained.

The full-length DNA of the present invention can be obtained by ligatingthe obtained cDNA fragments.

To determine the nucleotide sequence of the DNA obtained by the abovemethods, the DNA fragments or those cleaved by an appropriaterestriction enzyme(s) are introduced into a vector by standardtechniques, then the product is analyzed by a standard nucleotidesequence analysis technique, e.g., the dideoxy technique by Sanger etal. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or using nucleotidesequence analyzers of Perkin Elmer (373A.DNA sequencer), those ofPharmacia, and of LI-COR.

The DNA of interest can be prepared by chemical synthesis using a DNAsynthesizer based on the nucleotide sequence information obtained by theabove-mentioned methods. The DNA synthesizers include the onemanufactured by Shimazu Corp. using the thiophosfite technique, the one(model 392) by Perkin Elmer using the phosphoamidite technique, and thelike.

The novelty of the obtained nucleotide sequence can be confirmed bysearching a nucleotide sequence database of GenBank, EMBL, DDBJ and thelike, using a homology search program i.e., BLAST.

For a novel nucleotide sequence, after converting it to an amino acidsequence, an amino acid sequence database, e.g., GenPept, PIR, orSwiss-Prot, is searched using a homology search program e.g., FASTA, andFrame Search, thereby searching the existing genes having homologies.

The DNA of the present invention obtained by the above emthod can beexpressed in a host cell to produce the polypeptide of the presentinvention, according to the methods described in Molecular Cloning2^(nd) ed., Current protocols in Molecular Biology and the like.

That is, the polypeptide of the present invention can be produced byconstructing a recombinant vector wherein the DNA of the presentinvention is inserted an appropriate expression vector at an insertionsite located downstream of the promoter therein, transferring thisvector to a host cell to obtain a transformant expressing thepolypeptide of the present invention, and culturing this transformant.

As the host cells, any bacterial cells, yeast cells, animal cells,insect cells, plant cells and the like can be used, so long as thedesired gene can be expressed therein. Particularly, a transformantobtained by transferring the recombinant vector, in which the DNA of thepresent invention is inserted to introduce into a peripheral blood cellof a healthy individual, can be employed for the gene therapy ofHIV-infected individuals.

As the expression vectors, which are capable of autonomously replicatingin the host cell or being integrated into a chromosome and contain apromoter at a site appropriate for the transcription of the DNA of thepresent invention are used.

When a prokaryote cell such as a bacterial cell is used as the hostcell, the preferable recombinant vector expressing the polypeptide genewhich is a constituent of a T cell receptor of the present invention canautonomously replicate in the prokaryotes and is a recombinant vectorconsisting of a promotor, ribosome binding sequence, the DNA of thepresent invention, and a transcription termination sequence. The vectormay further comprise a gene requlating the promoter.

Examples of suitable expression vectors are pSE280 (manufactured byInvitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured byQIAGEN), pKYP10 (JP-A-58-110600), pKYP200[Agric. Biol. Chem., 48, 669(1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl.Acad. Sci., USA, 82, 4306 (1985)], pBluescript II SK(−) (STRATAGENE),pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2(FERM B-6798), pTerm2 (JP-A-3-22979, US4686191, US4939094, US5160735),pKK233-3 (manufactured by Amersham Pharmacia Biotech), pGEX(manufactured by Pharmacia), pET system (manufactured by Novagen),pSupex, pTrxFus (Invitrogen), and pMAL-c2 (New England Biolabs).

As the promoters, any promoters capable of being expressed in host cellscan be used. When Escherichia coli is used as a host, promoters derivedfrom such as Escherichia coli or phages include trp promotor (Ptrp), lacpromotor (Plac), P_(L) promoter, T7 promoter, P_(R) promoter and thelike. In addition, promoters, artificially designed and modified e.g.,Ptrp×2 formed by joining two Ptrp in series, tac promoter, T7lacpromoter, and let I promoter can be used. When Bacillus subtilis is usedas a host, the promoters include SP01 and SP02 that are phages ofBacillus subtilis, penP promoters, and the like.

As the ribosome binding sequence, a plasmid in which the distancebetween Shine-Dalgarno sequence and a starting codon is appropriatelyadjusted (e.g., 6 to 18 bases) can be used preferably.

A transcription termination sequence is not always necessary for theexpression of the DNA according to the present invention. Preferably,the transcription termination sequence is arranged directly after thestructural gene.

Examples of suitable host cells are cells of microorganisms belonging togenus Escherichia, genus Serratia, genus Bacillus, genus Brevibacterium,genus Coryneabacterium, genus Microbacterium, genus Pseudomonas, forexample, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue,Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276,Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101,Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49,Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratiamarcescens, Bacillus subtilis, Bacillus amyloliquefaciens,Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC14068,Brevibacterium saccharolyticum ATCC 14066, Corynebacterium glutamicumATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacteriumglutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870,Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110 and thelike.

Introduction of the recombinant vector can be carried out by any of themethod for introducing DNA into the above host cell, for example, themethod using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)],the protoplast method (JP-A-63-248394), and the electroporationmethod[Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111(1979)].

As the plasmid containing the DNA encoding the polypeptide, which is aconstituent of the killer T cell receptor of the present invention, forexample, pH-RT1α containing the DNA encoding the killer T cell receptorαchain or pH-RT1β containing the DNA encoding the killer T cell receptorβchain, or the like can be mentioend. Escherichia coli TG1/pH-RT1αcontaining the plasmid pH-RT1α and Escherichia coli TG1/pH-RT1βcontaining the plasmid pH-RT1β were deposited with National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology (1-3, Higashi-1-chome, Tsukuba-shi, Ibaraki-ken, Japan) asFERM BP-6078 and FERM BP-6079, respectively.

When a yeast cell is used as the host cell, YEp13 (ATCC37115), YEp24(ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like can be used asthe expression vectors.

As the promoter, any promoters capable of expressing in a yeast cell canbe used. Examples of suitable promoters are PH05 promoter, PGK promoter,GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shockpolypeptide promoter, MFα 1 promoter, and CUP 1 promoter.

The host cells can include yeast cells belonging to a genusSaccharomyces, genus Schizosaccharomyces, genus Kluyveromyces, genusTrichosporon, genus Schwanniomyces, genus Pichia, for exampleSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichiapastoris, and the like.

Introduction of the recombinant vector can be carried out by any of themethods for introducing DNA into yeast cells, for example theelectroporation [Methods in Enzymology, 194, 182 (1990)], thespheroplast method[Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)], and thelithium acetate method [Journal of Bacteriology, 153, 163 (1983)].

When an animal cell is used as a host cell, pcDNAI/Amp (manufactured byInvitrogen), pcDNAI, pAMoERC3Sc, pCDM8 [Nature, 329, 840 (1987)],pAGE107 [JP-A-3-22979, Cytotechnology, 3, 133 (1990)], pREP4(manufactured by Invitrogen), pAGE103 [Journal of Biochemistry, 101,1307 (1987)], pAMo, pAMoA, pAS3-3 (JP-A-2-227075) and the like can beused as the expression vector.

As the promotor, any promoters capable of expressing in animal cells canbe used. Example of suitable promoters are cytomegalovirus (CMV) IE(immediate early) gene promoter, SV40 initial promoter ormetallothionein promoter, retrovirus promoter, heat shock promoter, SR apromoter and the like. In addition, human CMV IE gene enhancer can beused with the promoter.

Examples of animal cells are mouse myeloma cells, rat myeloma cells,mouse hybridomas, human Namalwa cells, or Namalwa KJM-1 cells, humanfetal kidney cells, human leukocytes, African green monkey kidney cells,Chinese hamster CHO cells, HBT5637 (JP-A-63-299) and the like.

The mouse myeloma cells include SP2/0, NS0 and the like, the rat myelomacells include YB2/0 and the like, the human fetal kidney cells includeHEK293 (ATCC: CRL-1573) and the like, the human leukocytes includeBALL-1 and the like, and the African green monkey kidney cells includeCOS-1, COS-7 and the like.

Introduction of the recombinant vector can be carried out by any of themethods of introducing DNA into animal cells, for example theelectroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphatetransfection (JP-A-2-227075), and the lipofection method [Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)] and methods shown in Virology, 52, 456(1973) and the like.

When an insect cell is used as a host cell, the polypeptide can beexpressed by the methods described in Baculovirus Expression Vectors, ALaboratory Manual (W. H. Freeman and Company, New York (1992)),Molecular Biology, A Laboratory Manual, Current protocols in MolecularBiology, Bio/Technology, 6, 47 (1988) and the like.

That is, a recombinant vector for transferring a recombinant gene andbaculovirus are co-introduced into an insect cell to obtain arecombinant virus in the insect cell culture supernatant, then theinsect cell is infected with the recombinant virus, therefore thepolypeptide can be expressed.

Examples of the gene transfer vector suitable for use in this method arepVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).

An example of the Baculoviruses is Autographa californica nuclearpolyhedrosis virus, which is a virus infecting insects belonging tofamily Barathra.

Examples of the insect cells are the ovarian cells of Spodopterafrugiperda and of Trichoplusia ni, culture cells derived from a silkworm ovarium.

The ovarian cells of Spodoptera frugiperda include Sf9, Sf21(Baculovirus Expression Vectors, A Laboratory Manual) and the like,those of Trichoplusia ni include High 5, BTI-TN-5B1-4 (manufactured byInvitrogen) and the like, the culture cells from a silk worm ovariuminclude Bombyx mori N4 and the like.

Methods of transferring both said vector for transferring therecombinant gene and said baculovirus into an insect cell to prepare arecombinant virus include calcium phosphate transfection(JP-A-2-227075), lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)] and the like.

Methods of expressing genes include secretory production, fusion proteinexpression and the like according to the techniques shown in MolecularCloning 2^(nd) ed in addition to direct expression.

When the gene is expressed in yeast cell, an animal cell, or insectcells, a sugar or sugar chain-attached protein can be obtained.

The polypeptide that is a constituent of a T cell receptor of thepresent invention can be produced by culturing the transformant obtainedas described above to form the polypeptide that is a constituent of akiller T cell receptor of the present invention is formed, accumulatedin the culture, and recovering the polypeptide accumulated in theculture.

Further, the polypeptide, which is a constituent of a T cell receptor ofthe present invention, can be expressed in vivo by transferring theexpression vector to express the appropriate polypeptide, which is aconstituent of a T cell receptor of the present invention, into a cellcollected from a patient, and then by returning the cell into the body.

Culturing of the transformant of the present invention can be carriedout by conventional methods for culturing the host cell of thetransformant.

As the media for culturing of the transformant prepared by usingmicroorganisms such as Escherichia coli or yeasts as a host cell, any ofnatural media and synthetic media can be used insofar as it contains acarbon source, a nitrogen source, and inorganic salts, and the likewhich can be assimilated by the microorganism used, and the transformantis efficiently cultured therein.

As the carbon sources, any glucose, fructose, sucrose, molasses, starch,carbonhydrates such as hydrolysates of starch, organic acids e.g.,acetic acids and propionic acids, and alcohols e.g., ethanol andpropanol can be used.

As the nitrogen sources any ammonia, salts of inorganic acids or organicacids, such as ammonium chloride, ammonium sulfate, ammonium acetate,and ammonium phosphate, other substances nitrogen containing compounds,peptone, meat extract, yeast extract, corn steep liquor, caseinhydrolysates, soybean meal and soybean meal hydrolysate, variousfermentation microorganic cells or their digests, and the like can beused.

The inorganic substances used in the present invention include potassiumdihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate, calcium carbonate and the like.

Culturing is usually carried out under aerobic conditions, for example,by shaking cultures or submerged aeration stirring culture, at 15 to 40°C. for 16 to 96 hours. The pH is maintained at 3.0 to 9.0 during theculturing. The pH adjustment is carried out by using an inorganic ororganic acid, alkaline solution, urea, calcium carbonate, ammonia, andthe like.

If necessary, antibiotics such as ampicillin and tetracycline may beadded to the medium.

When a microorganism transformed with the expression vector comprisingan inducible promoter is cultured, an inducer may be added to the mediumif necessary. For example isopropyl-β-D-thiogalactopyranoside (IPTG) orthe like may be added in the case of microorganisms transformed with anexpression vector comprising lac promoter, and in the case ofmicroorganisms transformed with an expression vector comprising trppromoter, indoleacetic acid (IAA) or the like may be added.

For the culturing of the transformants prepared by using an animal cellas host cells include a generally used RPMI1640 media, Eagle MEM mediaor those to which fetal calf serum or the like is added may be used.Culturing is usually carried out in the presence of 5% CO₂ at 35 to 37°C. for 3 to 7 days. If necessary, antibiotics such as kanamycin andpenicillin may be added to the medium while culturing.

For the culturing of the transformant prepared by using an insect cellas the host cell, TNM-FH medium (manufactured by Pharmingen), Sf900 IISFM (manufactured by Life Technologies), ExCell1400 and ExCell405 (bothmanufactured by JRH Biosciences) and the like may be used.

Culturing is usually carried out at 25 to 30° C., at a pH ranging from 6to 7 and normally for 1 to 5 days. If necessary, antibiotics such asgentamicin may be added to the medium while culturing.

The polypeptide expressed in the above-described manner can be purifiedfrom the culture of the transformant by conventional methods forisolating and purifying enzymes to obtain the polypeptide which is aconstituent of T cell receptor of the present invention.

For example, when the polypeptide of the present invention is expressedin a soluble form within the cell, after the completion of culturing andthe cells are recovered by centrifugation, suspended in an aqueousbuffer, followed by disruption using an ultrasonic disruption, a Frenchpress, a Manton Gaulin homogenizer, a Dyno Mill, and the like to obtaina cell-free extract.

The cell-free extract is centrifuged, and from the obtained supernatant,a purified sample can be produced from the supernatant obtained bycentrifugation of the cell-free extract by conventional methods forisolating and purifying enzymes including a solvent extracting,salting-out with ammonium sulfate, desalting, precipitation with organicsolvents, anion exchange chromatography using resins such asdiethylaminoethyl (DEAE)—Sepharose and DIAION HPA-75 (manufactured byMitsubishi Chemical Corp.), cation exchange chromatography using resinse.g., S-Sepharose FF (manufactured by Pharmacia) and the like,hydrophobic chromatography using resins such as butyl sepharose, phenylsepharose and the like, gel filtration using molecular sieve, affinitychromatography, chromatofocusing, and electrophoresis such asisoelectric focusing, alone or in combination.

When the polypeptide is expressed in cells in an insoluble form, thecells are similarly disrupted, and separated by centrifugation, andfractions are precipitated, fraction. The polypeptide is recovered fromthe precipitate fraction by conventional method and the insolublepolypeptide is solubilized with a protein denaturing agent.

The solubilized solution is diluted or dialyzed to give a solutioncontaining no protein-denaturing agent or containing protein-denaturingagent at a low concentration so that proteins are not denatured and thenormal protein structure is restored, followed, by the same isolationand purification step as mentioned above to obtain a purified proteinpreparation.

When the polypeptide of the present invention or its derivatives such asa sugar-modified proteins are extracellularly secreted, the polypeptideor its derivatives such as the sugar chain-added from can be recoveredfrom the culture supernatant.

That is, the culture is treated by the above-described means such ascentrifugation, and the obtained soluble fractions is subjected to thesame isolation and purification methods as described above to obtain apurified sample.

Further, the polypeptide of the present invention can be produced as afusion protein with another protein and purified by affinitychromatography using substances having affinity for the fusion protein.For example according to the technique by Row et al., [Proc. Natl. Acad.Sci. USA, 86, 8227(1989), Genes Develop., 4, 1288 (1990)] or to methodsdescribed in JP-A-05-336963 and in JP-A-06-823021, the polypeptide ofthe present invention can be produced as a fusion protein with proteinA, and purified by affinity chromatography using immunoglobulin G.Moreover, the polypeptide of the present invention can be produced as afusion protein with a Flag peptide, and purified by affinitychromatography using an anti Flag antibody [Proc. Natl. Acad. Sci. USA,86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Furthermore, thepolypeptide of the present invention can be purified by affinitychromatography using an antibody specific to the polypeptide itself.

Moreover, the polypeptide of the present invention can be produced bychemical synthetic methods such as the Fmoc method (thefluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonylmethod) based on the amino acid sequence information contained in thepolypeptide.

Further, the peptide of the present invention can be chemicallysynthesized by using peptide synthesizers manufactured by AdvancedChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument,Synthecell-Vega, PerSeptive, Shimazu Corp., and the like.

The structural analysis for the purified polypeptide of the presentinvention can be carried out by methods conventionally used in ProteinChemistry, for example by techniques shown in Protein Structure Analysisfor Gene Cloning (Hisashi Hirano, Tokyo Kagaku Dojin, 1993).

The transgenic animals used herein means animals into which foreigngenes are introduced at their initial developmental stage. Thetransgenic animals include mice, rats, or livestock such as cattle andsheep. The transgenic mouse is prepared as described below.

The transgenic mouse of the present invention can be prepared accordingto the methods of Hogan, B. et al., [Manipulating the mouse embryo. Alaboratory manual. 2^(nd) ed. 1994. Cold Spring Harbor Laboratory Press,New York.] and Yamamura, K. et al., [J. Biochem., 9, 357-363 (1984)].That is, a female C57BL/6 mouse treated with a hormone is allowed tocross, and the fertilized ovum is taken out, a fragment of a gene to betransferred but having no part of a vector, which is prepared inadvance, is micro-injected using a micro-glass pipette into the malepronucleus of the fertilized ovum. Of the ova obtained to which thegenes are introduced, several hundreds of surviving ova are transplantedinto the uterine tubes of pseudo-pregnant mice, thereby generatingtransgenic mice. can be prepared as follows.

Animals are immunized using the proteins obtained by the above-mentionedmethod as antigens. For immunization the intact antigens may beadministered subcutatenously, intravenously, or intraperitoneally to theanimals. It is preferred to administer, the antigen in combination witha carrier protein with high antigenicity or an appropriate adjuvant.

The carrier proteins include Macroschisma sinense hemocyanin, Keyholelimpet hemocyanin, bovine serum albumin, bovine thyroglobulin and thelike. The adjuvants include complete Freund's adjuvant, alminiumhydroxide gel, pertussis vaccine and the like.

The animals for immunization include non-human mammals, including rats,goats, 3 to 20 weeks old mice, rats, hamsters and the like.

The antigen is administered 3 to 10 times every 1 to 2 weeks after thefirst administration. The dose of the antigen is preferably 50 to 100 μgper animal. On 3rd to 7th days after each administration, a blood sampleis collected from fundus oculi veniplex, and the obtained serum isexamined for reactivity to the antigen according to enzyme-linkedimmunosorbent assay [ELISA: IGAKU-SHOIN Ltd. (1976)] and the like.

Then non-human mammals, the serum of which shows a sufficient antibodytiter, are employed as a source for serum- or antibody-producing cells.

The polyclonal antibodies can be prepared by subjecting the serum toseparation and purification procedure.

The monoclonal antibody can be prepared by fusing the antibody-producingcells and a myeloma cells derived from a non-human mammal to obtainhybridoma, and culturing the obtained hydridoma or administering theobtained hybridoma to an animal to cause ascites tumor, and subjectingthe culture or the ascites to isolation and purification steps.

The antibody-producing cells are collected from splenic cells, the lymphnode, peripheral blood of a non-human mammal administered with theantigen.

As the myeloma cells, any myeloma cells capable of proliferating invitro can be used. Examples of suitable cells lines are 8-azaguanineresistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-U1(P3-Ul) [G.Kohler et al.,; Europ. J. Immunol., 6, 511 (1976)],SP2/0-Ag14(SP-2) [M. Shulman et al., ; Nature, 276, 269 (1978)],P3-X63-Ag8653(653) [J. F. Kearney et al.,; J. Immunol., 123, 1548(1979)], and P3-X63-Ag8(X63) [G.Kohler et al.,; Nature, 256, 495 (1975)]which is derived from a mouse. For culture or subculture of these cells,2×10⁷ or more of cells are secured before cell fusion according toAntibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, 1988(herein after abbreviated as A Laboratory Manual).

After the antibody producing cells obtained as described above and themyeloma cells are washed, a cell agglutination medium such aspolyethylene glycol-1000 (PEG-1000) is added to fuse these cells, andthen suspended in the medium. As the cell washing solution, examples ofthe solutions are an MEM medium, and a PBS (1.83 g of disodiumhydrogenphosphate, 0.21 g of potassium dihydrogenphosphate, 7.65 g ofsodium chloride, 1 l of distilled water, pH 7.2). As the medium used tosuspend fusion cells, examples of the media are a HAT medium, which isan normal medium (RPMI-1640 medium to which 1.5 mM of glutamine, 5×10⁻⁵M2-mercaptoethanol, 10 μg/ml of gentamicin and 10% fetal calf serum (FCS)(manufactured by CSL) are added) supplemented with 10⁻⁴M hypoxantine,1.5×10⁻⁵ M thymidine and 4×10⁻⁷M aminopterin, so that only the fusioncells of interest can be selectively obtained.

After the culturing, a portion of the culture supernatant is subjectedto enzyme immunoassay, to select cells which react with an antigenicprotein and do not react with an non-antigenic protein. Then cloning iscarried out by limiting dilution, and cells showing a high and stableantibody titer according to enzyme immunoassay are selected asmonoclonal antibody producing hybridoma cell lines.

Enzyme Immunoassay

Antigenic proteins or cells expressing antigenic proteins is coated on a96-well plate and allowed to react with a primary antibody, namely ahybridoma culture supernatant or a purified antibody.

After the primary antibody reaction, the plate is washed and a secondaryantibody are added.

The secondary antibody is an antibody obtained by labeling an antibody,which can recognize the immunoglobuline of the primary antibody with abiotin, an enzyme, a chemiluminescent substance, a radioactive compoundor the like. For example when a mouse is used to prepared hybriodmas, anantibody capable of recognizing the mouse immunoglobulin is used as thesecondary antibody.

After the above-mentioned reaction is finished, a reaction suitable fora substance labeling the secondary antibody is performed, therebyselecting hybriodmas that produce monoclonal antibodies specificallyreacting with the antigens.

The monoclonal antibodies can be prepared by separating and purifyingfrom the culture fluid obtained by culturing the hymbridomas; or fromthe ascites of the 8 to 10 week mice or nude mice, which are treatedwith 0.5 ml Pristane (2,6,10,14-tetramethylpentadecane) by administeringit intraperitoneally to the mice and are kept for 2 weeks, and to whichthe monoclonal antibody-producing hybridomas are administered so as tocause ascites tumor.

Monoclonal antibodies can be separated or purified by one or more of themethods including centrifugation, salting out using 40 to 50% saturatedammonium sulfate, caprylic acid precipitation method, chromatographiesusing DEAE-Sepharose column, anion exchange column, protein-A or -Gcolumn, or gel filtration column, and the like. The method allows torecover IgG or IgM fractions and obtain purified monoclonal antibodies.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of observation of which peptides are recognizedby established T cell clones, RT-1, RT-2 and RT-3.

FIG. 2 is the graph showing changes in cytotoxic activity when the cellderived from the established killer T cell clone RT-1 is treated with 1)only complement, 2) complement and anti-CD8 antibody, 3) complement andanti-CD4 antibody, and 4) the cell is not treated.

FIG. 3 shows the experimental results of studying: what type of class IMHC molecule is presented together with P18 for which the killer T cellclone RT-1 conduct specific recognization. Class I MHC:KDL means that inmice, class I MHC molecule presenting antigens comprises K, D, and Lregions. For example in mice B10.D2, class I MHC molecule comprisesK^(d), D^(d), and L^(d) regions.

FIG. 4 shows the result that V β 8.1 DNA obtained from the killer T cellclone RT-1 amplified by PCR was confirmed by agarose gel eletrophoresis.

FIG. 5 shows the result of analysis of the T-cell clone RT-1 stainedwith anti-β 8.1 antibody using flow cytometry.

FIG. 6 shows the result that V α 42H11 DNA obtained from the killer Tcell clone RT-1 amplified by PCR was confirmed by agarose geleletrophoresis.

FIG. 7 is the construction of the full-length cDNA for the RT-1 TCRαchain. For αchain, V α was screened using 5′ RACE method.

FIG. 8 is the construction of the full-length cDNA for RT-1 TCRβchain.For βchain, the PCR product of RT-1 junctional region was incorporatedinto βchain containing the known V β 8.1., using recombinant PCR.

FIG. 9 shows the expression vector BCMGSNeo for in vitro transfection ofRT-1TCRαchain and βchain.

FIG. 10 shows the expression vector pHSE3′ for transgenic of RT-1TCRαchain and βchain.

FIG. 11 shows the results of southern blot analysis for the transgenicmouse tail DNA of established TCRαchain and βchain.

FIG. 12 shows the results of analysis of the expression of transgenes ofRT-1 TCRαchain and βchain in a transgenic mouse. The expression ofTCRαchain was confirmed by RT-PCR since there is no antibodies toTCRαchain. The thymus and spleen were removed from the transgenic mouseto extract mRNA, then RT-PCR was performed. As a result, a bandcorresponding to TCRαchain was shown in both tissues (upper Figure). ForTCRβ chain, CD8⁺T cell was analyzed by FACS using anti-Vβ 8 (F23.1) as aspecific antibody (lower Figure).

FIG. 13 is the graph showing the specific cytotoxic activity of cellsderived from transgenic mice. LINE-OVA represents a T-cell line(negative control) reactive specifically with ovalbumin, LINE-IIIBrepresents a T-cell line (positive control) reactive specifically withHIV-IIIB strain, TG-spe (fresh/CD8 rich) is a spleen cell of thetransgenic mice and represents an uncultured CD8+cell, and TG-spe(fresh/whole) is a spleen cell of the transgenic mice and represents thewhole uncultured cell. As the target cells, Neo represents aNeo-gene-transferred BALB/c.3T3 cell (control cell), Neo*18MN representsa Neo-gene-transferred BALB/c.3T3 cell in which P18 peptide from HIV MNstrain was pulsed, and Neo*18IIIB represents a Neo-gene-transferredBALB/c.3T3 cell in which P18 peptide derived from HIV-IIIB strain waspulsed.

FIG. 14 is the graphs showing changes in the cytotoxic activity whencells derived from the transgenic mice were treated with 1) onlycomplements, 2) complements and anti-CD8 antibody, or 3) complements andanti-CD4 antibody. 15-12 represents transfectant, which is a BALB/c. 3T3cell into which HIV env gp160 gene was introduced, Neo*18IIIB representsa Neo-gene-transferred BALB/c.3T3 cell in which P18 peptide fromHIV-IIIB strain was pulsed, and Neo represents a Neo gene-transferredBALB/c.3T3 cell. In 2) it is shown that CD8⁺ T cell has a specifickiller activity because of disappearance of the cytotoxic activity bytreatment with the anti-CD8 antibody and complement.

FIG. 15 shows cytotoxic activity which is induced, by stimulation ofkiller T cells after that spleen cells of various transgenic mice werestimulated with the transfectant (15-12) which is a BALB/c.3T3 cell intowhich HIV env gene was introduced, with 15-12, Neo gene-transferredBALB/c.3T3 cell in which P18 peptide derived from HIV-IIIB strain waspulsed(Neo*18IIIB), or BALB/c.3T3 cell into which Neo gene wasintroduced (Neo control cell). Cytotoxic activity was observed not onlyin TCRαβ expressing transgenic mice, but also in TCRβ expressingtransgenic mice.

FIG. 16 shows the results of the study on α chain repertories (types)using RT-PCR before and after stimulation with HIV gp160 of TCRβexpressing transgenic mice having cytotoxic activity. Before thestimulation various type of the αchain were observed, but after thestimulation, a single type (V α 42H11J α 25) α chain was observed.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be illustrated in detail as follows, but itis contemplated that the scope of the present invention is not limitedthereto.

EXAMPLE 1

Establishment of P18 Specific Killer T Cell Clones (RT-1, RT-2, RT-3)

To establish P18 specific killer T cell clones (RT-1, RT-2, RT-3),BALB/c mice known to show high reactivity with P18 (H-2^(d) haplotype)(6-week old, female) [PNAS, 85, 3105 (1988)] were immunized byadministering via the tail vein recombinant vaccinia virus (10⁷PFU permouse) expressing a HIV-1 IIIB envelope protein gp160[Nature, 320, 535(1986)]. Four weeks later, the spleens were removed from the immunizedmice, and sensitized lymphocytes were prepared through steps includingthe removal of erythrocytes and the like. The sensitized lymphocyteswere stimulated by antigen-presenting cells of homotypic cell line,which P18 were bound to and which were irradiated (3,300 rad), or byP18-expressing fibroblast cells of homotypic cell line [PNAS, 85, 3105(1988)] (hereinafter referred to P18-expressing fibroblast), which isinactivated with mitomycin-C, thereby establishing a killer T cell linespecifically injuring P18-bound cells. Further, limiting dilution isperformed using a medium, RPMI1640 to which 10% FCS (fetal calf serum),2 mM L-glutamine, 100 U/ml penicillin, 10 μg/ml streptomycin, 5×10⁻⁵M2-mercaptoethanol, and 10 mM HEPES buffer were added [hereinafterreferred to as CTM (complete T cell medium)]. The medium containingkiller T cells was dispensed to wells of a round-bottomed 96-wellmicrotiter plate, each well containing 0.3 μl. The inactivatedfibroblast cells, 10⁴/well, were added to each well for re-stimulation.The half of the culture fluid was replaced with CTM containing 10% ratT-STIM (manufactured by Collaborative Research) every 3 to 4 days, andthe continuous stimulation using the P18-expressing fibroblast wasconducted once every two weeks, thereby to establish three P18 specifickiller T cell clones (RT-1, RT-2, RT-3) from about 1000 wells (FIG. 1).

FIG. 1 shows results of observation as to which peptides were presentedwhen various synthesized peptides (No. 1 to 55) covering HIV env wereused. The results indicate that RT-1, RT-2 and RT-3 specificallyrecognized the cell to which P18 corresponding to No. 18 was bound. Inaddition it was confirmed that all of clones were killer T cellsrestrained by CD8 molecule-positive D^(α) class I MHC molecule. Theresults are shown in FIGS. 2 and 3.

FIG. 2 is a graph showing the changes in cytotoxic activity when thecell derived from the established killer T cell clone RT-1 was treatedwith 1) only complements, 2) complements and anti-CD8 antibody, 3)complements and anti-CD4 antibody, or 4) the cell was not treated. Asshown in the graph, CD8⁺T cell obviously has a P18 specific killeractivity since the cytotoxic activity is removed by the treatment withthe complement and anti-CD8 antibody.

FIG. 3 shows results of study as to what type of class I MHC moleculesis presented with P18 for which the killer T cell clone RT-1 conductsthe specific recognition. In mice class I MHC molecules presentingantigens comprise K, D, and L regions. As shown in FIG. 3, the class IMHC molecules selectively recognize B10.D2 mouse that is restrained byD^(d) and have cytotoxic activity. Therefore, it is suggested that thekiller T cell clone RT-1 injures P18 by D^(d) class MHC's restraintability.

EXAMPLE 2

Isolation of T Cell Receptor Gene from P18-specific Killer T Cell Clone

V regions in P18-specific T cell receptor α- and β-chains weredetermined, followed by identification of gene sequences of each T cellreceptor region.

1. Extraction of mRNA from P18-specific Killer T Cell Clone RT-1

The established P18-specific killer T cell clone, RT-1, havingrelatively strong proliferation potency and killer activity amoung theestablished P18 specific killer T cell clones, could be increased to1×10⁸ cells for about 6 months by repeatedly stimulating as described inExample 1. To efficiently extract mRNAs from 5×10⁷ cell pellets, FastTrackVersion2.0 mRNA Isolation (manufactured by Invitrogen) was used. Asdescribed below in steps a) to g), mRNAs were extracted using oligo-dTcolumn from the lysate obtained by adding a surfactant agent.

a) Fifteen ml of a lysis buffer (which is 15 ml of stock buffer withinthe kit to which 0.3 ml of RNase protein degrader was added) was addedto the cell pellet transferred to a 50 ml tube, then the mixture wasstirred for 10 to 20 seconds.

b) The lysate obtained in a) was mixed using a 20 ml injection syringewith a 21 G needle, gently shaken in a thermostat at 45° C. for 60minutes, thereby decomposing proteins and RNA degrading enzymes.

c) Fifteen ml of the mixture obtained in b) to which 0.95 ml of 5M NaClwas added was stirred well. After that one oligo(dT) tablet, whichdirectly binds to mRNA and is attached to the kit, was put in thesolution, then it was gently shaken for 60 minutes at room temperature.

d) The mixture was centrifuged at 2000 rpm for 5 minutes, and thesupernatant was discarded. Twenty ml of a binding buffer within the kitwas added to the pellet, the suspension was washed by centrifugationseveral times at 2000 rpm for 5 minutes, and then applied to an oligo-dTcolumn.

e) The oligo-dT column to which 300 μl of low salt buffer was appliedwas repeatedly centrifuged at 5000 rpm for 10 seconds. After that atotal of 400 μl of elution buffer was applied to the oligo-dT column,then the solution was centrifuged at 5000 rpm for 10 seconds, therebyobtaining a mRNA extract.

f) Sixty μl of 2M sodium acetate and 1150 μl of 100% ethanol were addedto the mRNA extracts and stored in a freezer at −70° C. to −80° C. for aperiod of day and night.

g) An Eppendolf tube containing the mRNA pellet was centrifuged at 15000rpm for several seconds, then, 50 μl of elution buffer was added to thepellet. The absorbance was measured at 260 nm to calculate the quantityof mRNA extracted, followed by experiments for determining T cellreceptor sequences as shown below.

2. Determination of Type and Sequence of βChain

Unstable mRNA which is easily decomposed by RNase is easily converted tostable cDNA in the presence of reverse transcriptase and nucleic acidsas a substrate. Analysis as follows was performed using GeneAmp RNA PCRKit (manufactured by Perkin Elma Cetus) utilizing the above describedfact. V region in a mouse T cell receptor βchain (hereinafter referredto as V β region) is either one of V β1 to V β17. To amplify mRNAderived from the clone, RT-PCR was performed using a primer group thatwas designed based on a characteristic sequence at each 5′-end and aprimer (CB04E; SEQ ID NO: 2) that was designed based on a sequence of Cregion in the mouse T cell receptor (hereinafter referred to as Cβregion), common among all βchain. As a result of performing agarose geleletrophoresis for the obtained samples, cDNA amplification was seen forV β8 primer (SEQ ID NO:1). Subsequently, to identify the subclass of Vβ8, nested PCR was performed using a combination of V β8 subclass primerhaving a sequence different from the primer used as described above andCB04E primer (SEQ ID NO:2). Agarose gel eletrophoresis was performed forthe samples obtained so that amplification of cDNA was seen for V β8.1primer (SEQ ID NO: 3) (FIG. 4). Further, RT-1 was stained with an anti Vβ8.1 antibody (manufactured by Farmingene) and flow cytometry was used,thereby confirming that V region of expressed T cell receptor βchain wasβ8.1(FIG. 5). Moreover, DNAs were recovered and purified from the PCRproducts. To the DNAs, DyeDeoxy Terminator was added and electrophoresiswas performed. Then the T cell βchain gene sequence (SEQ ID NO: 6) wasdetermined using a gene sequence automatic analyzer ABI (manufactured byApplied Biosystem).

Detailed explanation will be given as follows.

Four μl of 25 mM MgCl₂ solution, 2 μl of PCR buffer (×10), 2 μl each ofdGTP, dATP, dTTP and dCTP, 1 μl of RNase inhibitor, 1 μl of reversetranscriptase, 1 μl of 3′-end primer (CB04E), and 2 μl of mRNA wereadded to a 0.5 ml microtube, and the solution was stirred using a voltexmixer for several seconds. After that one cycle of PCR (which consistsof 42° C. for 15 min., 99° C. for 5 min., and 5° C. for 5 min.) wasperformed. Next, 4 μl of MgCl₂, 2 μl of PCR buffer (×10), 65.5 μl ofdistilled water, and 0.5 μl of AmpliTaq DNA polymerase solution wereadded to the PCR reaction solution while 2 μl of 5′-end primers (V β1 toV β17) were added to each sample. One cycle (95° C. for 2 min.,), 35cycles (95° C. for 1 min., and 60° C. for 1 min.), and one cycle (5° C.for 7 min.) of PCR were performed.

Two% agarose gel eletrophoresis was performed for each of the obtainedsolutions corresponding to 5′-end primers (V β1 to V β17) so as toconfirm the presence or absence of bands. As a result, a DNA band wasconfirmed between V β8 (SEQ ID NO:1) primer and CB04E (SEQ ID NO:2)primer.

Next, the PCR reaction solution (50 μl to 100 μl in total) containingβchain cDNA, which is obtained using RT-1 mRNA derived from RT-1, primerfrom 5′ end of V β8.1 (SEQ ID NO:3), CB04E primer (SEQ ID NO:2) andreverse transcriptase was subjected to 1.0% agarose gel (SeaKem™ GTGAgarose) eletrophoresis followed by cutting the gel. The cut gel wasdissolved in sodium iodide (NaI) solution, to which glass powder forrecovering DNA (EASY TRAP TM Ver. 2, manufactured by Takara Shuzo Co.,Ltd.) was then added. The mixture was left for 5 minutes at roomtemperature so as to allow DNA to adsorb. Then the mixture was washedwith PBS, sterile distilled water or TE buffer was added to the pellet,and it was incubated at 55° C. for 5 minutes, thereby extracting DNA.Purified DNA in the supernatant was recovered, and the DNA gene sequencewas determined. Methods employed are as shown below.

The primer from 5′-end (SEQ ID NO:3) of V β8.1 and CB04E primer (SEQ IDNO:2), 3.2 pmol each, were added to about 50 to 200 ng of the recoveredDNA. Furthermore, deoxyribose, a terminator labeled with pigment andcontained in Ready Reaction DyeDeoxy Terminator Cycle Sequencing KitPRISM™ (manufactured by PERKIN ELMER CETUS), and AmpliTaq DNA polymeraseand H₂O were added to the DNA, and 25 cycles (where one cycle consistsof 96° C. for 10 seconds, 50° C. for 5 seconds 60° C. for 4 minutes) ofPCR were performed. The PCR product was applied to 6.75% Long Ranger™Gel (manufactured by Takara Shuzo Co., Ltd.), electrophoresis wasperformed with about 40 watt for 14 hours, and the result was read usinga gene sequence analyzer (ABI373 type, manufactured by AppliedBiosystem), thereby determining the entire gene sequence. As a result,the nucleotide sequence for T cell receptor βchain of RT-1 was V β8.1-Dβ-J β2.1-C β2. The amino acid sequence for T cell receptor βchain ofRT-1 was shown as SEQ ID NO: 7 and the nucleotide sequence as SEQ IDNO:6. Escherichia coli TG1/pH-RT1 β to which plasmid pH-RT1β containingDNA encoding T cell receptor βchain was transferred was deposited onAug. 26, 1997 with National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, Japan), and the assigned accessionnumber was FERM BP-6079.

3. Determination of Type and Sequence of a Chain

V regions in a mouse T cell receptor αchain (hereinafter referred to asV α region) containing 12 types of V regions ranging from V α1 to V α12and their subtypes are known to be more complex than βchain such thatthere are about 80 types of V regions. MRNA derived from clone RT-1 wasamplified by RT-PCR, as in the case for βchain, using a primer groupdesigned based on their characteristic sequences and a primer of C βregion, common among all αchains (exon-3 C α-R; SEQ ID NO:5) in the samemanner as in Example 2.2. However no amplification occurred for anyprimer though the experiment was repeated. Therefore, unusual many V αprimers were prepared based on database (GeneBank) and they were used toconfirm amplification with V α42H11 primer (SEQ ID NO: 4). Next, nestedPCR was performed using primers corresponding to various parts of Vα42H11. After amplification, electrophoresis was performed and thenbands were detected. Therefore it was confirmed that V α42H11 was aconstituent of RT-1 (FIG. 6).

As in the case for βchain, the αchain cDNA was prepared using mRNAderived from RT-1, V α42H11 (SEQ ID NO:4), exon-3C α-R(SEQ ID NO:5), andreverse transcriptase and purified, to which dideoxyribose labeled witha pigment was added, and the gene sequence was determined using a genesequence analyzer. As a result, the nucleotide sequence of T cellreceptor αchain of RT-1 was V α42H11-J α25-C α. The amino acid sequencefor T cell receptor αchain of RT-1 was shown as SEQ ID NO:9 and thenucleotide sequence as SEQ ID NO:8. Escherichia coli TG1/pH-RT1 α towhich plasmid pH-RT1 α containing DNA encoding T cell receptor αchainwas transferred was deposited on Aug. 26, 1997, with National Instituteof Bioscience and Human Technology, Agency of Industrial Science andTechnology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), andthe assigned accession number was FERM BP-6078.

EXAMPLE 3

Expression of T Cell Receptor and Functional Analysis

1. Preparation of Full-length cDNA of Clone RT-1 TCR αChain and βChain

The full-length cDNA clone was prepared to express the functional T cellreceptor as described below.

A. T Cell Receptor αChain

To obtain the full-length cDNA of αchain containing V α42H11 as shown inExample 2, DNA encoding VJC binding region (J region and parts of V andC regions at both ends of the J region) of the full-length cDNA derivedfrom T cell clone specific to insulin using known V α42H11 [Mol. Cell.Biol., 7, 1865-1872(1987)] was substituted for DNA encoding RT-1 VJCbinding region, thereby generating the full-length TCR αchain cDNAhaving a binding region specific to P18 (FIG. 7).

On the other hand, most T cell receptor αchains have several types ofsubfamilies in the identical V regions and a single T cell is known toexpress two αchains. Thus there are possibilities that V region sequencenear the VJC binding part detected by PCR is a different subfamily ofthe identical family, having a variant at its 5′ upstream even if it isidentical to V α42H11, or that the sequence expresses another totallydifferent αchain. Accordingly, cDNA at the upstream of C α was generatedfrom RT-1 mRNA by 5′ RACE method using a primer having an optionalsequence of C α site(GSP-1 and GSP-2 as shown in FIG. 7) and an oligo dTprimer, thereby determining the nucleotide sequence (SEQ ID NO:8). As aresult, it was shown that most clones obtained had nucleotide sequencesidentical to V α42H11 itself. However, minor clones were shown to haveαchain sequences that may be subfamilies (derived from insulin-specificT cell clone known as V α5.3.18) having a variation of two amino acidson the 5′ side of V α[Mol. Cell. Biol., 7, 1865-1872 (1987)]. Amino acidsequence of T cell receptor αchain of RT-1 of the minor clones was shownas SEQ ID NO: 11, and the nucleotide sequence as SEQ ID NO: 10. The twocan be considered to encode specific TCR αchain. Here, V α42H11 (aminoacid sequence: SEQ ID NO: 9, nucleotide sequence; SEQ ID NO: 8)consistent with the major clones was considered to be T cell receptorαchain V α region which is specific to P18. The V α42H11 was expressedas follows.

B. T Cell Receptor βChain

Unlike V α region, V β region has no subfamily. 5′ RACE method wasperformed without determining any sequence, V-D-J region and C regionwere amplified by RT-PCR and the resulting fragments were linkedtogether to obtain full-length V β8.1. DNA encoding VDJC binding region(D-J region and parts of V and C regions at both ends of the D-J region)of the full-length TCR βchain cDNA (p14 TCR β) of the T cell clonespecific to LCMV (lymphocytic choriomengitis virus) [EMBO J., 8, 719-727(1989)] that expresses V β8.1 was substituted for DNA encoding RT-1βchain VDJC binding region by recombinant PCR, thus generating thefull-length TCR β cDNA (FIG. 8). The amino acid sequence was shown inSEQ ID NO: 7, and the nucleotide sequence in SEQ ID NO: 6.

2. Generation of Expression Vectors for P18-specific T Cell Receptor αand βChain Genes

After confirming the full-length cDNA gene sequences for P18 specific Tcell receptor αchain (1.3 kb) and βchain (1.1 kb) both derived from RT-1(obtained in Example 3.1), each of them was inserted into an expressionvector. The expression vector BCMGS Neo [J.Exp. Med., 169, 13-25 (1989)]having a cytomegalovirus (CMV) promotor was employed for in vitrotransfection into a cell line (FIG. 9). The expression vector pHSE3′[EMBO J., 8, 719-727 (1989)] having H-2K^(b) promotor/Ig enhancer wasemployed for generating transgenic mice (FIG. 10). T cell receptorαchain (1.3 kb) and βchain (1.1 kb) were independently inserted at theXhoI site of the former vector, or inserted through blunt end ligationat BamH1/SalI sites of the latter vector, thus to generate recombinantvectors, BCMG-RT1 α, BCMG-RT1 β, pH-RT1 α and pH-RT1 β, respectively.

3. Transformation of RT-1 T Cell Receptor Gene into T Cell Line and invitro Expression of the Gene

BCMG-RT1 α and BCMG-RT1 β were transferred to a mutat T cell hybridomaTG40 [J. Immunol., 146, 3742-3746 (1991)], wherein gene coding for Tcell receptor α and βchains is deleted, by eletroporation. The T cellreceptor αβ requires CD8 as a conjugation receptor since it was derivedfrom killer T cell. Then, the expression vector BCMGSNeo into which CD8α and β genes were introduced was transferred to TG40 byelectroporation. The expression of T cell receptor complex was confirmedby FACS staining using antibodies (F23.1, 2C11)(manufactured byFarmingene) specific to V β8.1 and CD3 ε. As a result, clones havingboth the CD8 and the T cell receptor expressed therein were obtained.The functional expression of the prepared full-length RT-1 TCR α and βchains were strongly suggested because they were activated bystimulating with anti-T cell receptor antibodies and because they wereassociated with CD3 complex.

EXAMPLE 4

Transgenic Mice Expressing RT-1 T Cell Receptor

1. Preparation of Transgenic Mice Expressing T Cell Receptors

After excluding vector portions of pH-RT1 α and pH-RT1 β which wereindependently created by introducing, DNA encoding T cell receptorαchain and βchain (hereinafter referred to as TCR α-DNA, and as TCRβ-DNA, respectively) into pHSE3′, the DNAs were micro-injected intofertilized ova of C57BL/6 (H-2^(b)) mice, alone or in combination. Thatis in the first cycle, TCR α-DNA and TCR β-DNA were separately, and inthe second cycle, (TCR α-DNA) and (TCR β-DNA) together were injected.The tail DNA of mice born was prepared, and analyzed by PCR and Southernblotting. As shown in FIG. 11, it could be confirmed that transgenicmice in which TCR α and TCR β as transgenes were integrated,respectively, were obtained. These RT1TCR α- and RT1TCR β transgenicmice were crossed with wild type mice and further crossed with Balb/cmice. Since MHC genotype must be consistent with that of the originalRT-1 clone, RT1TCR α and RT1TCR β, both having H-2^(d) background, wereso crossed that mice expressing RT1TCR αβ and having H-2^(d) backgroundwere generated.

The expression of TCR α and β chains in the transgenic mice establishedas described above was examined. The results were shown in FIG. 12. ForTCR βchain, it was found by fluorescent staining using anti-V β8antibody (F23.1, manufactured by Farmingene) as described above thatmost of CD8 positive cells were V β8⁺ in the transgenic mice, though innormal mice V β8⁺ accounts for about 40%. On the other hand, theexpression of TCR αchain was analyzed by RT-PCR wherein mRNA wasexpressed using primers corresponding to the binding region since thereare no specific antibodies and staining cannot be performed. It wasshown that almost no RT-1 TCR αchain was detected in the thymus andspleen cells of the normal mice, but it was highly expressed in those ofthe transgenic mice.

2. Function of HIVgp160env-specific TCR-transgenic Mouse

Functions of the expressed TCR α and β were analyzed. The thymus andspleen cells were prepared from the mice expressing both TCR α and β,and their P18-specific cytotoxic activity was analyzed using anuntreated group and a group in which CD8 positive cells were enriched. Atransfectant into which P18 was previously transferred and a cell inwhich P18 was pulsed were used as target cells. As shown in FIG. 13, theresults suggested that when compared to a specific CTL line as apositive control, no specific killer activity was found even if CD8⁺cells were enriched among cells directly separated from the transgenicmice. However when this separated cell group was re-stimulated byco-culturing with a homotypic cell line that HIV-1 gp160 gene wasintroduced into and expressed in vitro, P18-specific killer activity wasobserved as shown in FIG. 14. Furthermore, since this activity wasremoved by treating with the anti-CD8 antibody and complement, it wasshown that CD8⁺ T cells bear specific killer activity. That is asexpected, RT-1TCR transgenic mice were shown to express killer T cellshaving HIVgp160-specific cytotoxic activity identical to that of theoriginal RT-1.

EXAMPLE 5

Induction of HIVgp160-specific Killer T Cells by HIVgp160specific-TCRβchain-transgenic Mice

The transgenic mice which have expressed only TCR βchain were analyzed.It has been considered that normally specific recognition is performedonly among TCR αβ-transgenic mice and no specific T cells are inducedfrom transgenic mice expressing TCR βchain only or TCR αchain onlybecause the recognition of antigens by T cells is performed by both TCRα and βchains. However as shown in FIG. 15, killer T cells specific top18 peptide were induced by separating the spleen cells of thetransgenic mice wherein HIVgp160-specific TCR βchain was expressed andby stimulating in vitro with cells expressing HIVgp160. Their antigenicspecificity was identical to the original killer T cell clone RT-1 fromwhich TCR was isolated. Then, the repertory of T cell TCR αchain inducedby in vitro stimulation was examined by RT-PCR. As shown in FIG. 16, theresults showed that before stimulation with HIVgp 160, T cell TCR αchainderived from TCR βchain-transgenic mice had random αchains, but afterstimulation most of CD8⁺T cells had TCR αchain completely consistentwith that of RT-1. That is in HIVgp160-specific T cells, uniformp18-psecific killer T cells having TCR α and βchains identical to thoseof RT-1 can be induced not only by stimulating T cells having both TCR αand βchains but also by stimulating T cells having TCR βchain only.

Industrial Applicability

The present invention provides a polypeptide which is a constituent of akiller T cell receptor injuring specifically human immunodeficiencyvirus-infected cells, a DNA encoding said polypeptide, a vectorcontaining said DNA, a transformant obtained by transforming with saidvector, a process for producing said polypeptide which is a constituentof the T cell receptor, transgenic animals having said polypeptideexpressed therein which is a constituent of the said killer T cellreceptor, and an antibody to said polypeptide. The polypeptide which isa constituent of the killer T cell receptor, can be useful as anti-HIVagents.

Sequence Listing Free Text

SEQ ID NO:1: An oligonucletoide synthesized based on the T cell receptorV β8 sequence.

SEQ ID NO:2: An oligonuceotide synthesized based on the CB04E sequence.

SEQ ID NO:3: An oligonucleotide synthesized based on the T cell receptorV β8.1 sequence.

SEQ ID NO:4: An oligonucletide synthesized based on the T cell receptorV a 42H11 sequence.

SEQ ID NO:5: An oligonucleotide synthesized based on the exon-3C α-Rsequence.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide based on thesequence of VYA8 of T cell receptor <400> SEQUENCE: 1 atatccctgatgggtacaag g 21 <210> SEQ ID NO 2 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Designed oligonucleotide based on the sequence of CB04E <400> SEQUENCE:2 ccgatgggag cacacgaacc cttaagc 27 <210> SEQ ID NO 3 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Designed oligonucleotide based on the sequence ofVYA8.1 of T cell receptor <400> SEQUENCE: 3 atgggctcca gactcttctt t 21<210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Designedoligonucleotide based on the sequence of VY“42H11 of T cell receptor<400> SEQUENCE: 4 atggactgtg tgtatgaaac 20 <210> SEQ ID NO 5 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide based on thesequence of exon-3CY”-R <400> SEQUENCE: 5 actggaccac agcctcagcg tc 22<210> SEQ ID NO 6 <211> LENGTH: 912 <212> TYPE: DNA <213> ORGANISM: Musmusculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(909)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(54) <221> NAME/KEY:mat_peptide <222> LOCATION: (55)..(909) <400> SEQUENCE: 6 atg ggc tccaga ctc ttc ttt gtg gtt ttg att ctc ctg tgt gca aaa 48 Met Gly Ser ArgLeu Phe Phe Val Val Leu Ile Leu Leu Cys Ala Lys -15 -10 -5 cac atg gaggct gca gtc acc caa agt cca aga agc aag gtg gca gta 96 His Met Glu AlaAla Val Thr Gln Ser Pro Arg Ser Lys Val Ala Val -1 1 5 10 aca gga ggaaag gtg aca ttg agc tgt cac cag act aat aac cat gac 144 Thr Gly Gly LysVal Thr Leu Ser Cys His Gln Thr Asn Asn His Asp 15 20 25 30 tat atg tactgg tat cgg cag gac acg ggg cat ggg ctg agg ctg atc 192 Tyr Met Tyr TrpTyr Arg Gln Asp Thr Gly His Gly Leu Arg Leu Ile 35 40 45 cat tac tca tatgtc gct gac agc acg gag aaa gga gat atc cct gat 240 His Tyr Ser Tyr ValAla Asp Ser Thr Glu Lys Gly Asp Ile Pro Asp 50 55 60 ggg tac aag gcc tccaga cca agc caa gag aat ttc tct ctc att ctg 288 Gly Tyr Lys Ala Ser ArgPro Ser Gln Glu Asn Phe Ser Leu Ile Leu 65 70 75 gag ttg gct tcc ctt tctcag aca gct gta tat ttc tgt gcc agc agt 336 Glu Leu Ala Ser Leu Ser GlnThr Ala Val Tyr Phe Cys Ala Ser Ser 80 85 90 gag ggg aga gag gct gag cagttc ttc gga cca ggg aca cga ctc acc 384 Glu Gly Arg Glu Ala Glu Gln PhePhe Gly Pro Gly Thr Arg Leu Thr 95 100 105 110 gtc cta gag gat ctg agaaat gtg act cca ccc aag gtc tcc ttg ttt 432 Val Leu Glu Asp Leu Arg AsnVal Thr Pro Pro Lys Val Ser Leu Phe 115 120 125 gag cca tca aaa gca gagatt gca aac aaa caa aag gct acc ctc gtg 480 Glu Pro Ser Lys Ala Glu IleAla Asn Lys Gln Lys Ala Thr Leu Val 130 135 140 tgc ttg gcc agg ggc ttcttc cct gac cac gtg gag ctg agc tgg tgg 528 Cys Leu Ala Arg Gly Phe PhePro Asp His Val Glu Leu Ser Trp Trp 145 150 155 gtg aat ggc aag gag gtccac agt ggg gtc agc acg gac cct cag gcc 576 Val Asn Gly Lys Glu Val HisSer Gly Val Ser Thr Asp Pro Gln Ala 160 165 170 tac aag gag agc aat tatagc tac tgc ctg agc agc cgc ctg agg gtc 624 Tyr Lys Glu Ser Asn Tyr SerTyr Cys Leu Ser Ser Arg Leu Arg Val 175 180 185 190 tct gct acc ttc tggcac aat cct cga aac cac ttc cgc tgc caa gtg 672 Ser Ala Thr Phe Trp HisAsn Pro Arg Asn His Phe Arg Cys Gln Val 195 200 205 cag ttc cat ggg ctttca gag gag gac aag tgg cca gag ggc tca ccc 720 Gln Phe His Gly Leu SerGlu Glu Asp Lys Trp Pro Glu Gly Ser Pro 210 215 220 aaa cct gtc aca cagaac atc agt gca gag gcc tgg ggc cga gca gac 768 Lys Pro Val Thr Gln AsnIle Ser Ala Glu Ala Trp Gly Arg Ala Asp 225 230 235 tgt gga atc act tcagca tcc tat cat cag ggg gtt ctg tct gca acc 816 Cys Gly Ile Thr Ser AlaSer Tyr His Gln Gly Val Leu Ser Ala Thr 240 245 250 atc ctc tat gag atccta ctg ggg aag gcc acc cta tat gct gtg ctg 864 Ile Leu Tyr Glu Ile LeuLeu Gly Lys Ala Thr Leu Tyr Ala Val Leu 255 260 265 270 gtc agt ggc ctggtg ctg atg gcc atg gtc aag aaa aaa aat tcc tga 912 Val Ser Gly Leu ValLeu Met Ala Met Val Lys Lys Lys Asn Ser 275 280 285 <210> SEQ ID NO 7<211> LENGTH: 303 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 7 Met Gly Ser Arg Leu Phe Phe Val Val Leu Ile Leu Leu Cys AlaLys -15 -10 -5 His Met Glu Ala Ala Val Thr Gln Ser Pro Arg Ser Lys ValAla Val -1 1 5 10 Thr Gly Gly Lys Val Thr Leu Ser Cys His Gln Thr AsnAsn His Asp 15 20 25 30 Tyr Met Tyr Trp Tyr Arg Gln Asp Thr Gly His GlyLeu Arg Leu Ile 35 40 45 His Tyr Ser Tyr Val Ala Asp Ser Thr Glu Lys GlyAsp Ile Pro Asp 50 55 60 Gly Tyr Lys Ala Ser Arg Pro Ser Gln Glu Asn PheSer Leu Ile Leu 65 70 75 Glu Leu Ala Ser Leu Ser Gln Thr Ala Val Tyr PheCys Ala Ser Ser 80 85 90 Glu Gly Arg Glu Ala Glu Gln Phe Phe Gly Pro GlyThr Arg Leu Thr 95 100 105 110 Val Leu Glu Asp Leu Arg Asn Val Thr ProPro Lys Val Ser Leu Phe 115 120 125 Glu Pro Ser Lys Ala Glu Ile Ala AsnLys Gln Lys Ala Thr Leu Val 130 135 140 Cys Leu Ala Arg Gly Phe Phe ProAsp His Val Glu Leu Ser Trp Trp 145 150 155 Val Asn Gly Lys Glu Val HisSer Gly Val Ser Thr Asp Pro Gln Ala 160 165 170 Tyr Lys Glu Ser Asn TyrSer Tyr Cys Leu Ser Ser Arg Leu Arg Val 175 180 185 190 Ser Ala Thr PheTrp His Asn Pro Arg Asn His Phe Arg Cys Gln Val 195 200 205 Gln Phe HisGly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 210 215 220 Lys ProVal Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp 225 230 235 CysGly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr 240 245 250Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 255 260265 270 Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser 275280 285 <210> SEQ ID NO 8 <211> LENGTH: 825 <212> TYPE: DNA <213>ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(822) <221> NAME/KEY: sig_peptide <222> LOCATION:(1)..(63) <221> NAME/KEY: mat_peptide <222> LOCATION: (64)..(822) <400>SEQUENCE: 8 atg ctg att cta agc ctg ttg gga gca gcc ttt ggc tcc att tgtttt 48 Met Leu Ile Leu Ser Leu Leu Gly Ala Ala Phe Gly Ser Ile Cys Phe-20 -15 -10 gca gca acc agc atg gcc cag aag gta aca cag act cag act tcaatt 96 Ala Ala Thr Ser Met Ala Gln Lys Val Thr Gln Thr Gln Thr Ser Ile-5 -1 1 5 10 tct gtg gtg gag aag aca acg gtg aca atg gac tgt gtg tat gaaacc 144 Ser Val Val Glu Lys Thr Thr Val Thr Met Asp Cys Val Tyr Glu Thr15 20 25 cgg gac agt tct tac ttc tta ttc tgg tac aag caa aca gca agt ggg192 Arg Asp Ser Ser Tyr Phe Leu Phe Trp Tyr Lys Gln Thr Ala Ser Gly 3035 40 gaa ata gtt ttc ctt att cgt cag gac tct tac aaa aag gaa aat gca240 Glu Ile Val Phe Leu Ile Arg Gln Asp Ser Tyr Lys Lys Glu Asn Ala 4550 55 aca gtg ggt cat tat tct ctg aac ttt cag aag cca aaa agt tcc atc288 Thr Val Gly His Tyr Ser Leu Asn Phe Gln Lys Pro Lys Ser Ser Ile 6065 70 75 gga ctc atc atc acc gcc aca cag att gag gac tca gca gta tat ttc336 Gly Leu Ile Ile Thr Ala Thr Gln Ile Glu Asp Ser Ala Val Tyr Phe 8085 90 tgt gct atg aga gag gat ggg ggc agt ggc aac aag ctc atc ttt gga384 Cys Ala Met Arg Glu Asp Gly Gly Ser Gly Asn Lys Leu Ile Phe Gly 95100 105 act ggc act ctg ctt tct gtc aag cca aac atc cag aac cca gaa cct432 Thr Gly Thr Leu Leu Ser Val Lys Pro Asn Ile Gln Asn Pro Glu Pro 110115 120 gct gtg tac cag tta aaa gat cct cgg tct cag gac agc acc ctc tgc480 Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys 125130 135 ctg ttc acc gac ttt gac tcc caa atc aat gtg ccg aaa acc atg gaa528 Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu 140145 150 155 tct gga acg ttc atc act gac aaa act gtg ctg gac atg aaa gctatg 576 Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met160 165 170 gat tcc aag agc aat ggg gcc att gcc tgg agc aac cag aca agcttc 624 Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe175 180 185 acc tgc caa gat atc ttc aaa gag acc aac gcc acc tac ccc agttca 672 Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser190 195 200 gac gtt ccc tgt gat gcc acg ttg act gag aaa agc ttt gaa acagat 720 Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp205 210 215 atg aac cta aac ttt caa aac ctg tca gtt atg gga ctc cga atcctc 768 Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu220 225 230 235 ctg ctg aaa gta gcg gga ttt aac ctg ctc atg acg ctg aggctg tgg 816 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg LeuTrp 240 245 250 tcc agt tga 825 Ser Ser <210> SEQ ID NO 9 <211> LENGTH:274 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 9 MetLeu Ile Leu Ser Leu Leu Gly Ala Ala Phe Gly Ser Ile Cys Phe -20 -15 -10Ala Ala Thr Ser Met Ala Gln Lys Val Thr Gln Thr Gln Thr Ser Ile -5 -1 15 10 Ser Val Val Glu Lys Thr Thr Val Thr Met Asp Cys Val Tyr Glu Thr 1520 25 Arg Asp Ser Ser Tyr Phe Leu Phe Trp Tyr Lys Gln Thr Ala Ser Gly 3035 40 Glu Ile Val Phe Leu Ile Arg Gln Asp Ser Tyr Lys Lys Glu Asn Ala 4550 55 Thr Val Gly His Tyr Ser Leu Asn Phe Gln Lys Pro Lys Ser Ser Ile 6065 70 75 Gly Leu Ile Ile Thr Ala Thr Gln Ile Glu Asp Ser Ala Val Tyr Phe80 85 90 Cys Ala Met Arg Glu Asp Gly Gly Ser Gly Asn Lys Leu Ile Phe Gly95 100 105 Thr Gly Thr Leu Leu Ser Val Lys Pro Asn Ile Gln Asn Pro GluPro 110 115 120 Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser ThrLeu Cys 125 130 135 Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro LysThr Met Glu 140 145 150 155 Ser Gly Thr Phe Ile Thr Asp Lys Thr Val LeuAsp Met Lys Ala Met 160 165 170 Asp Ser Lys Ser Asn Gly Ala Ile Ala TrpSer Asn Gln Thr Ser Phe 175 180 185 Thr Cys Gln Asp Ile Phe Lys Glu ThrAsn Ala Thr Tyr Pro Ser Ser 190 195 200 Asp Val Pro Cys Asp Ala Thr LeuThr Glu Lys Ser Phe Glu Thr Asp 205 210 215 Met Asn Leu Asn Phe Gln AsnLeu Ser Val Met Gly Leu Arg Ile Leu 220 225 230 235 Leu Leu Lys Val AlaGly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 240 245 250 Ser Ser <210>SEQ ID NO 10 <211> LENGTH: 822 <212> TYPE: DNA <213> ORGANISM: Musmusculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(819)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(63) <221> NAME/KEY:mat_peptide <222> LOCATION: (61)..(819) <400> SEQUENCE: 10 atg ctg attcta agc ctg ttg gga gca gcc ttt ggc tcc att tgt ttt 48 Met Leu Ile LeuSer Leu Leu Gly Ala Ala Phe Gly Ser Ile Cys Phe -20 -15 -10 -5 gca accagc atg gcc cag aag gta aca cag act cag act tca att tct 96 Ala Thr SerMet Ala Gln Lys Val Thr Gln Thr Gln Thr Ser Ile Ser -1 1 5 10 gtg atggag aag aca acg gtg aca atg gac tgt gtg tat gaa acc cag 144 Val Met GluLys Thr Thr Val Thr Met Asp Cys Val Tyr Glu Thr Gln 15 20 25 gac agt tcttac ttc tta ttc tgg tac aag caa aca gca agt ggg gaa 192 Asp Ser Ser TyrPhe Leu Phe Trp Tyr Lys Gln Thr Ala Ser Gly Glu 30 35 40 ata gtt ttc cttatt cgt cag gac tct tac aaa aag gaa aat gca aca 240 Ile Val Phe Leu IleArg Gln Asp Ser Tyr Lys Lys Glu Asn Ala Thr 45 50 55 60 gtg ggt cat tattct ctg aac ttt cag aag cca aaa agt tcc atc gga 288 Val Gly His Tyr SerLeu Asn Phe Gln Lys Pro Lys Ser Ser Ile Gly 65 70 75 ctc atc atc acc gccaca cag att gag gac tca gca gta tat ttc tgt 336 Leu Ile Ile Thr Ala ThrGln Ile Glu Asp Ser Ala Val Tyr Phe Cys 80 85 90 gct atg aga gag gat gggggc agt ggc aac aag ctc atc ttt gga act 384 Ala Met Arg Glu Asp Gly GlySer Gly Asn Lys Leu Ile Phe Gly Thr 95 100 105 ggc act ctg ctt tct gtcaag cca aac atc cag aac cca gaa cct gct 432 Gly Thr Leu Leu Ser Val LysPro Asn Ile Gln Asn Pro Glu Pro Ala 110 115 120 gtg tac cag tta aaa gatcct cgg tct cag gac agc acc ctc tgc ctg 480 Val Tyr Gln Leu Lys Asp ProArg Ser Gln Asp Ser Thr Leu Cys Leu 125 130 135 140 ttc acc gac ttt gactcc caa atc aat gtg ccg aaa acc atg gaa tct 528 Phe Thr Asp Phe Asp SerGln Ile Asn Val Pro Lys Thr Met Glu Ser 145 150 155 gga acg ttc atc actgac aaa act gtg ctg gac atg aaa gct atg gat 576 Gly Thr Phe Ile Thr AspLys Thr Val Leu Asp Met Lys Ala Met Asp 160 165 170 tcc aag agc aat ggggcc att gcc tgg agc aac cag aca agc ttc acc 624 Ser Lys Ser Asn Gly AlaIle Ala Trp Ser Asn Gln Thr Ser Phe Thr 175 180 185 tgc caa gat atc ttcaaa gag acc aac gcc acc tac ccc agt tca gac 672 Cys Gln Asp Ile Phe LysGlu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 190 195 200 gtt ccc tgt gat gccacg ttg act gag aaa agc ttt gaa aca gat atg 720 Val Pro Cys Asp Ala ThrLeu Thr Glu Lys Ser Phe Glu Thr Asp Met 205 210 215 220 aac cta aac tttcaa aac ctg tca gtt atg gga ctc cga atc ctc ctg 768 Asn Leu Asn Phe GlnAsn Leu Ser Val Met Gly Leu Arg Ile Leu Leu 225 230 235 ctg aaa gta gccgga ttt aac ctg ctc atg acg ctg agg ctg tgg tcc 816 Leu Lys Val Ala GlyPhe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 240 245 250 agt tga 822 Ser<210> SEQ ID NO 11 <211> LENGTH: 273 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 11 Met Leu Ile Leu Ser Leu Leu Gly Ala Ala PheGly Ser Ile Cys Phe -20 -15 -10 -5 Ala Thr Ser Met Ala Gln Lys Val ThrGln Thr Gln Thr Ser Ile Ser -1 1 5 10 Val Met Glu Lys Thr Thr Val ThrMet Asp Cys Val Tyr Glu Thr Gln 15 20 25 Asp Ser Ser Tyr Phe Leu Phe TrpTyr Lys Gln Thr Ala Ser Gly Glu 30 35 40 Ile Val Phe Leu Ile Arg Gln AspSer Tyr Lys Lys Glu Asn Ala Thr 45 50 55 60 Val Gly His Tyr Ser Leu AsnPhe Gln Lys Pro Lys Ser Ser Ile Gly 65 70 75 Leu Ile Ile Thr Ala Thr GlnIle Glu Asp Ser Ala Val Tyr Phe Cys 80 85 90 Ala Met Arg Glu Asp Gly GlySer Gly Asn Lys Leu Ile Phe Gly Thr 95 100 105 Gly Thr Leu Leu Ser ValLys Pro Asn Ile Gln Asn Pro Glu Pro Ala 110 115 120 Val Tyr Gln Leu LysAsp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu 125 130 135 140 Phe Thr AspPhe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 145 150 155 Gly ThrPhe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp 160 165 170 SerLys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 175 180 185Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 190 195200 Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met 205210 215 220 Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile LeuLeu 225 230 235 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg LeuTrp Ser 240 245 250 Ser

What is claimed is:
 1. A polypeptide which is a constituent of a killerT cell receptor and is capable of specifically recognizing humanimmunodeficiency virus envelope protein gp
 160. 2. The polypeptideaccording to claim 1 wherein the recognition region of the killer T cellreceptor which specifically recognizes human immunodeficiency virusenvelope protein gp160 is gp160V3 region.
 3. The polypeptide accordingto claim 2, wherein the recognition region is a region comprising aminoacid sequence 315 to 329 of V3 region of human immunodeficiency virusenvelope protein gp160.
 4. A polypeptide which comprises an amino acidsequence shown in SEQ ID NO: 7 or 9, or a polypeptide which comprises anamino acid sequence wherein one or more of amino acids in the amino acidsequence are substituted, deleted or added, and is capable ofspecifically recognizing human immunodeficiency virus envelope proteingp
 160. 5. A DNA encoding the polypeptide according to claim 1 or
 4. 6.A DNA having a nucleotide sequence shown in SEQ ID NO: 6 or
 8. 7. Aprocess for producing the polypeptide according to claim 1 or 4, whichcomprises culturing on a medium a transformant comprising a host cellharboring a vector comprising DNA encoding said polypeptide, forming andaccumulating the polypeptide in the culture, and then recovering thepolypeptide from the culture.
 8. An antibody which specifically reactedwith the polypeptide according to claim 1 or
 4. 9. The polypeptideaccording to claim 1 or 4, having a human type constant region site. 10.Transgenic animals having the polypeptide according to claim 1 or 4expressed therein.
 11. Anti-HIV agents containing the polypeptideaccording to claim 1 or
 4. 12. A DNA which encodes a polypeptide,capable of recognizing specifically human immunodeficiency virusenvelope protein gp160, which can hybridize with a DNA encoding thepolypeptide according to claim 1 under stringent conditions.
 13. A DNAwhich encodes a polypeptide, capable of recognizing specifically humanimmunodeficiency virus envelope protein gp 160, which can hybridize witha DNA encoding the polypeptide according to claim 4 under stringentconditions.
 14. A recombinant vector comprising the DNA according toclaim 5 and a vector.
 15. A recombinant vector comprising the DNAaccording to claim 6 and a vector.
 16. A recombinant vector comprisingthe DNA according to claim 12 and a vector.
 17. A recombinant vectorcomprising the DNA according to claim 13 and a vector.
 18. Atransformant obtained by introducing the recombinant vector according toclaim 14 into a host cell.
 19. A transformant obtained by introducingthe recombinant vector according to claim 15 into a host cell.
 20. Atransformant obtained by introducing the recombinant vector according toclaim 16 into a host cell.
 21. A transformant obtained by introducingthe recombinant vector according to claim 17 into a host cell.
 22. Amethod for treating HIV-infected individuals comprising administering atherapeutically effective amount of the transformant according to claim18 to a patient.
 23. A method for treating HIV-infected individualscomprising administering a therapeutically effective amount of thetransformant according to claim 19 to a patient.
 24. A method fortreating HIV-infected individuals comprising administering atherapeutically effective amount of the transformant according to claim20 to a patient.
 25. A method for treating HIV-infected individualscomprising administering a therapeutically effective amount of thetransformant according to claim 21 to a patient.